Our overall goal is to identify and characterize new molecular mechanisms of integrin biological function and intracellular signaling. Integrin receptors and the extracellular matrix proteins to which they bind play crucial roles in a wide variety of cellular interactions that are important in human development, health, and disease. Integrins mediate or regulate cell adhesion, migration, and extracellular matrix assembly, as well as cell surface control of the cytoskeleton, gene expression, and growth. We are addressing the following general questions focused on integrin-matrix interactions: 1. How do integrins, the extracellular matrix, and the cytoskeleton interact to produce cell migration? 2. What integrin signaling pathways function in particular biological functions such as migration? 3. How do cells assemble a fibronectin-based extracellular matrix? 4. What differs between signaling and biology in traditional two-dimensional (2D) cell culture and three-dimensional (3D) matrices characteristic of in vivo microenvironments? We are using a variety of cell and molecular biology approaches to address these questions, including biochemical analyses, fluorescent chimeras, fluorescence time-lapse microscopy, and mutational analysis. We have generated a variety of fluorescent molecular chimeras and/or mutants of cytoskeletal proteins, including focal adhesion kinase (FAK), vinculin, paxillin, and tensin, in an ongoing program to analyze their functions in integrin-mediated processes. Our particular focus this past fiscal year has been on integrin functions in cell migration and matrix assembly. The previous fiscal year, we had shown that an evolutionarily conserved site in the beta-1 integrin cytoplasmic tail (tryptophan 775) can regulate a specific signaling function, the phosphorylation of Akt. We subsequently found that this mutant and its low Akt activity result in low levels of active Rac, a critical regulator of the cytoskeleton and cell migration. Cells expressing this mutated integrin migrated in anomalously straight paths with a high degree of persistence in one direction. We then extended this initial finding to search for a general role for Rac activation in regulating the directionality of cell migration. In various human cell types, both normal and malignant including primary fibroblasts and an epithelial cell line, experimental manipulation of Rac activity using RNA interference by siRNA demonstrated that the total level of Rac activity serves as a molecular switch between random and directional cell migration. A relatively small reduction of 30% in total Rac activity switched cells to intrinsically directional migration, i.e. persistence of motility in one direction. The cell biological mechanism involves regulation by the level of active Rac of the number of peripheral lamellae, which are locomotory structures that give cells alternative choices for directions of migration. Cells with reduced total Rac activity had few peripheral lamellae but continued to use a single leading lamella for persistent forward migration. We also established that the mechanism of regulation of directionality by Rac activity was distinct from the lipid signaling system used in chemotaxis. Rac levels therefore provide a novel molecular switch between random and directionally persistent migration for a variety of cell types. We had previously characterized molecular machinery mediating integrin-based assembly of a fibronectin matrix. We collaborated with the Humphries laboratory in England to characterize a novel monoclonal antibody they had developed that identifies a specific epitope on the major fibronectin receptor alpha5-beta1. We showed that a particular molecular conformation promotes the characteristic linear directional translocation of integrin receptors on the cell surface needed to organize fibronectin molecules into extracellular fibrils in human fibroblasts and salivary epithelial cells. In living cells, this antibody locked the fibronectin receptor into a conformation promoting fibronectin matrix assembly. The existence of this unique conformation indicates that integrins are not merely on or off in terms of function, but that they can also adopt a particular conformation that is characteristic of, and promotes, fibronectin matrix assembly. A particularly well-studied mediator of integrin cytoskeletal and signaling interactions is the scaffold protein termed focal adhesion kinase (FAK). We had previously published a series of studies on FAK concerning its roles in adhesion and migration in normal and tumor cells. We collaborated with two groups studying other roles of FAK. In ongoing studies with the Takino laboratory in Japan, FAK was found to function cooperatively with the recently described protein JSAP1, which serves as a signaling scaffold for the MAP kinase JNK. This intermolecular cooperation between major signaling pathway components stimulated cell migration. Yet another pathway implicated in cell migration by normal and malignant cells is the MMP (matrix metalloproteinase) system. FAK was shown in a second collaboration with the Cid laboratory in Spain to regulate the release of MMP-2 and MMP-9 by human T cells. A novel finding was that different regions of FAK have distinct regulatory functions in this process, identifying new complexity in its downstream regulatory functions. Our studies have required the development of new methods for the quantification of fibronectin matrix assembly and for experimentally regulating the levels of active Rho family GTPases such as Rac, so we have provided detailed protocols in two methods papers. We also continue to provide unique research materials to many extramural researchers. For example, during this fiscal year, we completed dozens of formal NIH material transfer agreements to provide the research tools we have developed to researchers worldwide. In summary, our Section is elucidating mechanisms by which integrins function in cell adhesion, migration, and matrix assembly. Such analyses are important for understanding how these major adhesion receptors control cell and tissue movements, organization, growth, and development. We will continue to search for novel mechanisms and modulators. Knowledge of these basic processes should facilitate creative approaches to therapy, particularly in the fields of tissue engineering and cancer biology.